SUMMARY

The reasons why many insects breathe discontinuously at rest are poorly
understood and hotly debated. Three adaptive hypotheses attempt to explain the
significance of these discontinuous gas exchange cycles (DGCs), whether it be
to save water, to facilitate gas exchange in underground environments or to
limit oxidative damage. Comparative studies favour the water saving hypothesis
and mechanistic studies are equivocal but no study has examined the
acclimation responses of adult insects chronically exposed to a range of
respiratory environments. The present research is the first manipulative study
of such chronic exposure to take a strong-inference approach to evaluating the
competing hypotheses according to the explicit predictions stemming from them.
Adult cockroaches (Nauphoeta cinerea) were chronically exposed to
various treatments of different respiratory gas compositions (O2,
CO2 and humidity) and the DGC responses were interpreted in light
of the a priori predictions stemming from the competing hypotheses.
Rates of mass loss during respirometry were also measured for animals
acclimated to a range of humidity conditions. The results refute the
hypotheses of oxidative damage and underground gas exchange, and provide
evidence supporting the hypothesis that DGCs serve to reduce respiratory water
loss: cockroaches exposed to low humidity conditions exchange respiratory
gases for shorter durations during each DGC and showed lower rates of body
mass loss during respirometry than cockroaches exposed to high humidity
conditions.

INTRODUCTION

Since Heller's observation (Heller,
1930), the discontinuous gas exchange cycles (DGCs) exhibited by
many quiescent tracheated arthropods have proven to be a source of intrigue
and great debate. Insect DGCs are distinguished from continuous and cyclic
breathing patterns by regular periods where respiratory gas exchange is
essentially prevented due to spiracular closure
(Marais and Chown, 2003).
Typically, DGCs comprise three phases: closed (C), flutter (F) and open (O),
and the patterns of respiratory gas exchange occurring during these cycles has
been extensively described in lepidopteran pupae (e.g.
Hetz and Bradley, 2005;
Levy and Schneiderman, 1966a;
Levy and Schneiderman, 1966b;
Terblanche et al., 2008).
During the C phase the spiracles are tightly occluded and gas exchange with
the atmosphere is essentially prevented. Pressure within the tracheae declines
as CO2 is buffered within the haemolymph and O2 is
depleted due to respiration. Once the partial pressure of oxygen
(PO2) within the tracheal system has declined
to ∼2–4 kPa, the F phase is initiated. During this phase the
spiracles open and close with high frequency, facilitating inward convective
movement of air, such that a low and stable PO2
is maintained within the tracheal system
(Hetz and Bradley, 2005;
Levy and Schneiderman, 1966a).
Outward movement of H2O and CO2 is minimised as a result
of the inward convective movement of air, and CO2 continues to be
buffered in the haemolymph (Wobschall and
Hetz, 2004). When the partial pressure of CO2
(PCO2) within the tracheal system reaches ∼5–6
kPa, the spiracles open and respiratory gases are exchanged with the
atmosphere. CO2 is expelled in a burst and O2 moves
inwards until intratracheal PCO2 reaches ∼3–4
kPa and the cycle is repeated (Levy and
Schneiderman, 1966a).

DGCs are observed in a range of arthropod species
(Klok et al., 2002) and are
present in at least five insect orders. Species exhibiting DGCs inhabit xeric,
mesic, subterranean and non-subterranean environments, and are both winged and
wingless. The presence of DGCs in phylogenetically independent groups of
insects (Blattodea, Orthoptera, Coleoptera, Ledpidoptera and Hymenoptera)
suggests that the breathing pattern is adaptively significant, rather than
exists as an ancestral trait (Marais et
al., 2005). Three main hypotheses have emerged that attempt to
explain the adaptive significance of DGCs
(Chown et al., 2006). The
hygric hypothesis follows the original suggestions of Buck, Keister and Specht
(Buck et al., 1953) that DGCs
reduce transpiratory water loss. The chthonic hypothesis
(Lighton, 1998) postulates
that DGCs are an adaptation to facilitate efficient gas exchange under hypoxic
and/or hypercapnic conditions, often characteristic of underground
environments. Lighton and Berrigan
(Lighton and Berrigan, 1995)
originally proposed this hypothesis in combination with the hygric hypothesis,
such that DGCs serve to facilitate gas exchange in challenging conditions
whilst also avoiding respiratory water loss. In recent literature, however,
the pure chthonic hypothesis, irrespective of water loss, has become prominent
(Chown et al., 2006). The
final hypothesis is the oxidative damage hypothesis
(Bradley, 2000), which suggests
that DGCs function to limit oxidative damage to tissues. Because the trachae
are capable of rapidly delivering oxygen when required (i.e. during flight),
when at rest, near-ambient levels of oxygen at the ends of the tracheoles may
potentially be harmful to the insects' tissues.

To date, research examining the adaptive function of DGCs has not been well
integrated. A mixture of mechanistic and comparative studies fails to provide
unequivocal support for any of the current hypotheses. One possible approach
for investigating the function of DGCs involves analysing potential changes in
the insects' gas exchange patterns in response to environmental variation.
Many organisms can respond to changes in the environment through morphological
or physiological alterations that allow improved function in the new
conditions. This process of change in response to environmental variation is
known as phenotypic plasticity or acclimation response
(Fordyce, 2006). Until now,
during examination of DGCs, adult insects have only been subjected to acute
changes in respiratory gas conditions, as opposed to being chronically
exposed. It has therefore not yet been discovered whether or not insects are
capable of modifying their gas exchange patterns in response to prolonged
changes in respiratory environments. The potential acclimation response of an
insect to a range of environmental conditions could be utilised to
differentiate among the three putative adaptive functions of DGCs, as each
hypothesis can be used to make distinct predictions regarding the changes in
DGC patterns in response to different respiratory environments
(Table 1). The literature is
largely devoid of prediction-based approaches for understanding the function
of DGCs, and such a strong-inference approach would give more credibility to
results (Huey et al., 1999).
The present research is the first manipulative strong-inference study to
address changes in the DGCs of adult insects in response to chronic exposure
to varying respiratory environments. This research makes it possible to
differentiate among the competing hypotheses and provides insight into the
possible selective pressures that may have led to the evolution of DGCs by
evaluating the hypotheses according to the explicit predictions stemming from
them.

The present study aimed to test among the competing hypotheses for the
function of DGCs using the speckled cockroach (Nauphoeta cinerea).
Cockroaches were chronically exposed to different concentrations of
O2, CO2 and water vapour [in practice relative humidity
(RH)] and DGC responses were examined in light of the a priori
predictions of the competing hypotheses
(Table 1). In the case of the
hygric hypothesis, a positive relationship between O phase duration and RH
treatment is predicted, as most respiratory water loss occurs during the O
phase (Chown et al., 2006).
Thus, animals exposed to low levels of ambient RH will have shorter O phases
than animals acclimated to high RH. In the case of the chthonic hypothesis,
either a positive relationship between CO2 treatment and the C and
F phase durations or a negative relationship between O2 treatment
and the C and F phase durations is predicted, because the CO2 and
O2 partial pressure gradients required to facilitate efficient gas
exchange are generated during these phases. Thus, animals acclimated to low
O2, high CO2 or both are predicted to have relatively
long C and F phases, such that large partial pressure gradients are
established to maintain adequate gas exchange under hypoxic or hypercapnic
conditions. Finally, in the case of the oxidative damage hypothesis, a
negative relationship between O phase duration and O2 treatment is
predicted, because oxidative damage would be greatest during the O phase.
Thus, animals exposed to high O2 are predicted to have shorter O
durations that animals acclimated to low O2.

MATERIALS AND METHODS

Nauphoeta cinerea Olivier 1789 was a suitable study organism for
this research as, following preliminary investigations, it was shown to
exhibit a conspicuous DGC (Fig.
1). Final instar cockroaches were obtained from The Herp Shop
(Ardeer, Victoria, Australia) and maintained as single-sex stock populations
in 60 l plastic containers at a constant temperature of 23±1.5°C
and a 12 h:12 h L:D cycle. Cockroaches were provided with an ad
libitum diet of carrots and dry cat food. The stock population was
maintained at environmental conditions: 21% O2, 0.03% (atmospheric)
CO2 and ambient RH (∼60–80%). Upon maturation, samples of
male cockroaches from the stock population were randomly selected and assigned
to acclimation treatments. Females were not used in this study to eliminate
changes in metabolism and gas exchange associated with reproduction
(Rossolimo, 1982), as female
N. cinerea are facultatively parthenogenetic
(Corely et al., 2001).

In order to elucidate whether or not DGC patterns showed an acclimation
response, cockroaches were chronically exposed to a number of different gas
conditions. Exposure treatments lasted five weeks, a period adequate to elicit
acclimation responses in cockroaches
(Dehnel and Segal, 1956). For
each of the gases [O2, CO2 and water vapour (RH)], a
range of treatments from low to high was used. Each treatment population
(N∼50) was housed in a 7 l polypropylene (Sistema, New Zealand)
container under the same temperature and L:D conditions as the stock
population. The treatment gases were set and delivered to the acclimation
boxes at a flow rate of ∼200 ml min–1, measured with a
mechanical flow meter (Duff and McIntosh, Sydney, Australia). This ensured
constant turnover of the gas within the container and maintained a slight
positive pressure inside the container. Gas exited the container via
a minimum of 1 m of 8 mm outer diameter tubing.

To ascertain whether a change in DGC pattern occurred during the exposure
period, cockroach respiratory patterns were characterised at 23±1°C
upon completion of acclimation treatments. As such, the rate of CO2
release of 12–16 randomly selected cockroaches was measured using
standard flow-through respirometry
(Withers, 2001). Two
cockroaches were measured simultaneously using each of the two sample cells of
a Li-7000 (Li-Cor, Nebraska, USA) CO2–H2O
analyser. This precluded simultaneous measurement of CO2 and
H2O but increased the number of individuals that could be measured.
Cockroaches were placed individually in one of two 25 ml respirometry chambers
to which gas (see Table 2 and
below for details) was delivered at a constant flow rate of 200 ml
min–1. Unless explicitly stated otherwise, the incurrent gas
was dry (Drierite, Sigma-Aldrich, Steinheim, Germany) and CO2-free
(Soda Lime, Fluka, Steinheim, Germany) to maximise the accuracy of the
analyser. The fractional CO2 content of the excurrent gas from each
chamber was recorded to a computer at a sampling frequency of 1 Hz.

Measurement of carbon dioxide release over time from Nauphoeta
cinerea, demonstrating a conspicuous discontinuous gas exchange cycle.
Measurements were taken at a flow rate of 200 ml min–1.

All respirometry was performed during the inactive phase of the circadian
cycle (daytime), and food was withdrawn at least 24 h prior to measurements.
After being placed in the respirometry chamber, cockroaches were allocated a
one-hour `settling in' period. The gas exchange patterns of the animals were
then measured under the appropriate gases, which were presented sequentially
in a random order during a single respirometry session. The chamber was
darkened to encourage resting behaviour (and hence initiation of DGCs). The
mass of each cockroach was also recorded to 0.001 g before and after
respirometry measurements.

Oxygen exposure comprised four treatments: 5, 10, 21 and 40±1.1%
O2, and carbon dioxide and relative humidity each comprised three
treatments (0.03, 3±0.03 and 6±0.3% CO2, and
25±0.1, 45±0.3 and 90±1.4% RH, respectively). Compressed
mixes of O2, CO2 and N2 obtained from and
certified by a commercial supplier (BOC gases, Brisbane, Australia) were used
for the O2 and CO2 acclimations. Desired levels of RH
were produced by equilibrating saturated air with water vapour at a range of
temperatures (2, 10 and 21°C for 25, 45 and 90% RH at 23°C) using
constant temperature cabinets, and were verified using a RH-300 Water Vapour
Analyser (Sable Systems, Las Vegas, NV, USA).
Table 2 provides an overview of
the nominal levels of acclimation treatments (`acclimation gas' hereafter) and
the gas conditions under which DGCs were measured (`measurement gas'
hereafter). Following acclimation treatments, cockroaches were measured under
the conditions to which they were chronically exposed, as well as under the
conditions of the other treatments for a particular gas where possible. Thus,
animals acclimated to 5, 10, 21 or 40% were measured at each of these
O2 concentrations in dry air, animals acclimated to 0, 3 or 6%
CO2 were measured at 0% CO2 in dry air (measurement at
higher levels of CO2 was not possible because the analyser
saturated at 50 p.p.m. CO2), and animals acclimated to 25, 45 or
90% RH were measured at 25 and 45% RH (due to the risk of condensation in the
analyser at 90% RH).

The recorded data were used to characterise respiratory gas exchange
patterns in Microsoft Excel (Redmond, WA, USA), and only individuals
exhibiting DGCs were used for analysis. For each DGC, total DGC (O+CF), O and
CF phase durations were recorded and metabolic rates were calculated according
to Withers (Withers, 2001):
where V̇CO2=rate
of CO2 production,
V̇I=carbon dioxide
concentration, FeCO2=excurrent fraction of
CO2 and RE=respiratory exchange ratio, which was assumed
to be 0.8. Rate of CO2 production was used as a proxy for metabolic
rate.

C and F phases were combined due to the difficulty of unambiguously
differentiating the F phase in all individuals, and because F phase may
commence before CO2 release is detected using flow-through
respirometry (Hadley and Quinlan,
1993; Harrison et al.,
1995; Wobschall and Hetz,
2004). Mixed model analysis of variance (ANOVA) and analysis of
covariance (ANCOVA) were used to test for an effect of acclimation treatment
on total DGC, O and CF phase durations. The individual identification number
of cockroaches was included as a random effect to account for the measurement
of multiple cycles per individual, and in the cases of O2 and RH,
to account for the measurement of individuals in multiple gas conditions. In
initial analyses, the following variables were included: acclimation
treatment, time (am or pm), chamber, resting (settling in) gas, measurement
gas, measurement order, mass, metabolic rate and identification number. In
subsequent analyses, non-significant variables were eliminated and any
significant variables were analysed for an interaction with acclimation
treatment. Final models always included acclimation treatment, measurement
gas, mass, metabolic rate and identification number regardless of their
significance. An interaction between acclimation treatment and measurement gas
was always tested for, and any other significant covariates or interactions
were also included. Data were tested for normality using Shapiro–Wilk
tests, and non-normal data were transformed to improve normality
(log10 or square root). In one quarter of the cases, data did not
reach normality. In these circumstances the transformation that rendered the
data closest to normal distribution was accepted, as according to the Central
Limit Theorem, the distribution of means tends toward normality for large
sample sizes despite a non-normal population distribution
(Quinn and Keough, 2002;
Zar, 1974).

Additionally, to determine if RH acclimation had an effect on water loss,
rates of mass loss during respirometry were compared for animals acclimated to
25, 45 and 90% RH using ANCOVA with body mass as a covariate. All statistical
tests were conducted using JMP v.7.0.1 (SAS Institute Inc., Cary, NC, USA),
and α was set at 0.05 for all tests. For clarity, adjusted means are
presented in figures, and are shown ±s.e.m.

RESULTS

The effect of acclimation treatment on DGC duration is always reported
regardless of significance. There was never a significant interaction between
acclimation treatment and measurement gas (P>0.05 in all cases).
Other covariates and interactions are only reported if their effects were
significant, except in cases where a significant covariate did not have a
significant interaction with acclimation treatment, in which case the
non-significant interactions are also reported. In addition,
Table 3 provides a summary of
the mean initial mass and mean metabolic rates for each acclimation treatment
at the conclusion of the chronic exposure period.

A summary of mean initial mass and mean metabolic rate of cockroaches in
each acclimation treatment

Carbon dioxide

Mass had a significant effect on total DGC duration (ANOVA
F1,32=7.6, P=0.01) but there was no significant
interaction between mass and CO2 acclimation treatment (ANOVA
F2,26=0.84, P=0.44). There was a significant
effect of acclimation treatment on total DGC duration (ANOVA
F2,29=7.52, P=0.002), and 6% CO2
exposure resulted in significantly shorter DGC durations compared with 0% and
3% (Tukey's HSD) (Fig. 2).

There was a significant effect of mass and acclimation treatment on O phase
duration (ANOVA F1,29=4.58, P=0.04;
F2,27=8.03, P=0.002) but there was no significant
interaction between mass and treatment (ANOVA F2,24=0.56,
P=0.58). O phase duration was significantly shorter following 3% and
6% CO2 treatments when compared with 0% (Tukey's HSD)
(Fig. 2).

There was a significant effect of acclimation treatment on CF phase
duration (ANOVA F2,30=6.7, P=0.004). CF phase
duration was significantly shorter following exposure to 6% CO2
than following exposure to 3%, and neither were significantly different from
0% (Tukey's HSD) (Fig. 2).

Relative humidity

Metabolic rate had a significant effect on total DGC duration (ANOVA
F1,138=6.8, P=0.01) but there was no significant
interaction between metabolic rate and RH acclimation treatments (ANOVA
F2,118=2.7, P=0.07). There was a significant
effect of treatment (ANOVA F2,24=6.1, P=0.007),
with exposure to 90% RH resulting in significantly longer total DGC duration
compared with 25% (Tukey's HSD) (Fig.
3).

There was no effect of metabolic rate on O phase duration, so metabolic
rate was excluded from subsequent analyses of O phase. There was a significant
effect of acclimation treatment on O phase duration (ANOVA
F2,23=8.9, P=0.001). O phase duration was
significantly longer following exposure to 90% compared with 25% RH (Tukey's
HSD) (Fig. 3).

Adjusted least square means of total discontinuous gas exchange cycles
(DGC), open (O) and closed-flutter (CF) phase durations following exposure to
relative humidity (RH) treatments, shown +s.e.m. Exposure to 90% RH resulted
in longer DGC and O durations than exposure to lower levels of RH. Total
sample size: N=184 measurements from 27 individuals (25%:
N=72 measurements from 10 individuals; 45%: N=38
measurements from 6 individuals; 90%: N=70 measurements from 10
individuals). Columns within a phase that share a letter (A or B) are not
significantly different from each other (Tukey's HSD).

Oxygen

Initial analyses revealed that O2 acclimation treatment,
measurement gas and measurement order had significant effects on total DGC
duration (ANOVA F3,66=4.2, P=0.008;
F3,431=13.0, P<0.0001;
F3,424=4.6, P=0.004). There was also a
significant interaction between treatment and measurement order (ANOVA
F9,420=3.3, P=0.0007).

There was no effect of acclimation treatment on CF phase duration (ANOVA
F3,65=0.38, P=0.77) but there was a significant
interaction between acclimation treatment and measurement order (ANOVA
F9,420=2.1, P=0.03).

Acclimation treatment had a significant effect on O phase duration (ANOVA
F3,60=16.9, P=0.0001) but there was a significant
interaction between acclimation treatment and metabolic rate, and between
acclimation treatment and measurement order (ANOVA
F1,218=23.6, P<0.0001;
F3,217=4.3, P=0.006, respectively). Exploratory
examination of these effects suggested a difference in acclimation response
between hypoxic and hyperoxic conditions, and subsequent analyses were
conducted on hypoxic (5, 10 and 21% O2, measured at each of these
levels) and hyperoxic (21 and 40% O2, measured at each of these
levels) groups separately.

Hypoxic group

Acclimation treatment had a significant effect on total DGC duration (ANOVA
F2,49=5.4, P=0.007) and there was a significant
interaction between treatment and measurement order (ANOVA
F6,247=2.6, P=0.02). Only the 21% treatment
measured in the first hour was significantly different from that measured in
the third hour (Tukey's HSD) (Fig.
5A).

Mean rates of mass loss for the three relative humidity (RH) treatments,
shown +s.e.m. Exposure to 25% RH (N=10) resulted in a decreased rate
of mass loss compared with mass loss rates following exposure to 45 and 90% RH
(N=6 and 10, respectively). Columns that share a letter (A or B) are
not significantly different from each other (Tukey's HSD).

Both acclimation treatment and measurement gas had a significant effect on
O phase duration (ANOVA F2,40=7.1, P=0.002;
F2,236=50.8, P<0.0001, respectively) and there
was a significant interaction between acclimation treatment and measurement
order (ANOVA F6,237=3.1, P=0.006). Only the 21%
treatment measured in the first hour was significantly different to the
measurement in the second hour (Tukey's HSD)
(Fig. 5B).

DISCUSSION

The present research is the first of its kind to demonstrate that adult
insects alter their respiratory gas exchange patterns in response to chronic
exposure to varying environments. Cockroaches showed a significant acclimation
response to each of the O2, CO2 and RH treatments. These
responses are compared with the explicit predictions based on the three
adaptive hypotheses in order to elicit support for any number of these
hypotheses.

Adjusted least square means of phase durations in the hypoxic group of
O2 treatments according to measurement order (hour), shown +s.e.m.
A positive relationship between acclimation and total discontinuous gas
exchange cycles (DGC) and open (O) phase durations is apparent but only in the
first two hours of measurement (A and B). There was no effect of acclimation
treatment on the closed-flutter (CF) phase duration (C). Total sample size:
N=275 measurements from 34 individuals (5%: N=69
measurements from 8 individuals; 10%: N=81 measurements from 11
individuals; 21%: N=125 measurements from 15 individuals). Columns
that share a letter (A or B) are not significantly different from each other
(Tukey's HSD).

Chthonic hypothesis

CF phase duration was shortest following exposure to high levels of
CO2 and longer when exposed to lower levels
(Fig. 2). This response runs
counter to the predictions set out by the chthonic hypothesis, which suggests
that C and F phase duration will increase in hypercapnia to facilitate
adequate gas exchange via a steep respiratory gas gradient.
Similarly, there was no significant effect of O2 acclimation
treatments on C and F phase duration. This further refutes the chthonic
hypothesis, which proposes an increase in the C and F duration as
O2 levels decrease, again to facilitate adequate gas exchange.
Unfortunately, however, we were only able to measure animals in conditions of
0% CO2, and so it remains unknown how animals acclimated to high
levels of CO2 will exchange respiratory gases in hypercapnia. The
few species that have been measured have been shown to cease DGCs in
hypercapnia (Harrison et al.,
1995; Terblanche et al.,
2008), and it would be interesting to determine if this is also
the case for cockroaches acclimated to hypercapnia.

Oxidative damage hypothesis

The significant interaction between acclimation and measurement order
demonstrates that the effects of oxygen acclimation are dependent on
measurement order. Although Tukey's HSD does not identify any significant
pair-wise differences between O2 treatments
(Fig. 5A,B), there is an
apparent positive relationship between hypoxic O2 treatments and
the DGC and O phase durations. This relationship is only apparent in the first
one to two hours of measurement (i.e. the second and third hours of total
respirometry session), after which it appears to be obscured. Nevertheless,
regardless of whether O phase duration increases with O2 treatments
or remains unchanged, both responses are clearly inconsistent with the
predictions made by the oxidative damage hypothesis, which states that the O
phase should decrease in duration following exposure to higher levels of
oxygen in order to protect tissues from oxidative damage. The lack of an
acclimation response to hyperoxia further suggests that DGCs are not required
to limit oxidative damage. Similarly, although intratracheal
PO2 is regulated at 4–5 kPa during the CF
phase in atmospheres of up to 50 kPa O2 in atlas moths and silkworm
pupae (Hetz and Bradley, 2005;
Levy and Schneiderman, 1966a),
this regulation is not maintained at higher
PO2s (Levy
and Schneiderman, 1966b).

Hygric hypothesis

The hygric hypothesis recently received support from work by Marais et al.
(Marais et al., 2005) and
White et al. (White et al.,
2007) and the present research lends further credence to the
original explanation for the adaptive function of DGCs
(Buck et al., 1953;
Buck and Keister, 1955;
Burkett and Schneiderman,
1974a; Kestler,
1985; Levy and Schneiderman,
1966a; Lighton,
1990; Lighton et al.,
1993). Exposure to low levels of RH results in a reduction in DGC
duration, as well as a reduction in the duration of the O phase whereas the
duration of the CF phase was unaffected
(Fig. 3). The change in O
duration is consistent with the explicit predictions that stem from the hygric
hypothesis (Table 1). O phase
durations were longest following acclimation to high levels of humidity where
the saturation deficit between the respiratory surfaces and that atmosphere is
likely to be small, and rates of water loss are likely to be low. Following
exposure to low humidity, O phase durations were shorter, which presumably
acted to reduce respiratory water loss. This finding is further supported by
the fact that mean rates of mass loss were 5–10-fold higher following
acclimation to 45 and 90% RH treatments than when compared with 25% RH
acclimation (Fig. 4). It is
acknowledged that mass loss alone is not a definitive measure of respiratory
water loss as it does not discriminate between mass lost via
defecation, cuticular or respiratory transpiration. Further work examining
only respiratory water loss would provide an improved point of comparison.

Given that cockroaches show an acclimation response to altered ambient
humidity, it is surprising that measurement humidity did not have a
significant effect on phase durations. Cockroaches therefore appear unable to
detect acute changes in ambient humidity. It is possible that the acute
exposure is too short a time for an observable response to occur but it is
nevertheless clear that cockroaches do not respond to RH immediately as they
do to changes in O2 and CO2. Potentially, cockroaches
chronically exposed to low levels of humidity have lower levels of body
hydration than those chronically exposed to high levels of humidity. Thus, the
acclimation response to humidity may actually represent a response to varying
levels of hydration. Such a desiccated state is likely to alter the haemolymph
PCO2 and pH (Chown,
2002), leading to a change in ventilation rate
(Snyder et al., 1980).
However, while DGC frequency does increase following acclimation to low RH, CF
phase duration remains unchanged. If desiccation-associated changes in
haemolymph pH were responsible for the acclimation response to RH, one might
expect to see a decrease in the CF phase duration as internal CO2
would reach the O phase trigger more quickly, because the volume, and
therefore presumably the CO2 buffering capacity, of the haemolymph
is reduced. This however is not what is observed for the CF phase duration.
Alternatively, the level or concentration of buffers could increase as a
consequence of desiccation, and therefore total buffer capacity would remain
constant, in which case CF duration would be expected to be independent of
hydration status. Clearly, chronic exposure to varying levels of ambient
humidity offers exciting opportunities to gain further insight into the
mechanistic basis of DGCs. At this stage, however, the mechanism by which
cockroaches sense and respond to altered humidity remains unclear.

Conclusion

The present study has answered calls in the literature for a
single-species, strong-inference manipulative approach to examine the
evolutionary significance of DGCs (Chown,
2002; Chown et al.,
2006; Lighton,
2007; Lighton and Turner,
2008; Marais et al.,
2005; Quinlan and Gibbs,
2006; Terblanche et al.,
2008). The present research provides support for the hygric
hypothesis and disputes both the chthonic and oxidative damage hypotheses.
This is in contrast with a recent study by Terblanche et al., which provided
support for the oxidative damage hypothesis and limited support for the hygric
hypothesis (Terblanche et al.,
2008). Terblanche et al. exposed diapausing moth pupae Samia
cynthia to a range of levels of O2, CO2 and
humidity and interpreted the responses of the animals in light of the explicit
predictions of the competing hypotheses
(Terblanche et al., 2008).
However, Terblanche et al. examined only the effect of acute exposure on
immature insects (Terblanche et al.,
2008). It is well documented that low O2 and high
CO2 levels cause insect spiracles to open
(Beckel and Schneiderman, 1957;
Burkett and Schneiderman, 1967;
Burkett and Schneiderman,
1974b), so it is to be expected that DGCs will cease in hypoxia or
hypercapnia. Studies that only examine the DGC responses of insects to acutely
altered levels of respiratory gases are therefore of limited value when
distinguishing between the various hypotheses for the evolution of DGCs.

Testing among the predictions that stem from the three adaptive hypotheses
explaining the evolution of DGCs demonstrates a clear support for the hygric
hypothesis. However, implicit in this approach is the assumption that the best
model is included in the candidate set
(Johnson and Omland, 2004;
Quinn and Dunham, 1983). It
remains to be seen whether future studies continue to find support for water
loss as the driving force for the evolution and maintenance of discontinuous
ventilation or whether new hypotheses need to be considered. It has also been
suggested that several factors are likely to work together to influence the
expression of DGCs (Chown,
2002). It would be advantageous if further research were to be
conducted examining the effect on DGCs of combinations of the gas conditions
reported here. Such an approach would aid in revealing possible interactive
effects of the gas variables that may not be detected when variables are
examined in isolation. Indeed in reality, insects encounter microclimates of
low O2 and high CO2 rather than the individually
manipulated gas variable of the present study
(Anderson and Ultsch, 1987).
Furthermore, the level of intratracheal O2 influences the level of
CO2 at which spiracles open, and vice versa
(Burkett and Schneiderman,
1967; Burkett and Schneiderman,
1974b), so it is possible that a combination of hyperoxia and
hypercapnia may elicit acclimation responses different to those observed in
the present study. Such an approach could also reveal whether or not DGC
expression is prioritised according to the most costly variable in the
immediate respiratory environment. For example, for animals experiencing water
loss stress, DGCs may become important in terms of the oxidative damage
hypothesis. Wigglesworth (Wigglesworth,
1935) documented the presence of fluid in the ends of the tracheae
under hyperoxic conditions and Kestler
(Kestler, 1985) proposed that
this fluid functioned to restrict tracheal conductance and hence decrease
potential damage resulting from high levels of O2. If animals
become dehydrated, they may be unable to fill the tracheae with water, leaving
them vulnerable to oxidative damage. In such instances, O2 levels
may become an important factor for the exhibition of DGCs.

The support garnered for the hygric hypothesis from the research presented
here suggests that reducing respiratory water loss was a significant factor in
the evolution of DGCs, at least in N. cinerea. The hygric hypothesis
is the first of the three adaptive hypotheses to be supported by a variety of
studies: two broad scale comparative studies
(Marais et al., 2005;
White et al., 2007) that
examined a wide range of species from a diverse range of habitats, many
mechanistic studies dealing with acute exposures of respiratory gases (e.g.
Chown and Davis, 2003;
Duncan et al., 2002a;
Duncan et al., 2002b;
Duncan and Dickman, 2001;
Lighton et al., 1993), and now
a mechanistic study that has examined the effect of chronic exposure to
various respiratory environments. A thorough understanding of the evolution of
physiological traits and their ecological implications is aided by the
strength of a number of complementary approaches such as these
(Huey and Kingsolver, 1993).
Nevertheless, it is important that further studies of acclimation to chronic
exposure are conducted on a variety of species, particularly from other orders
(such as Hymenoptera, Lepidoptera, Orthoptera and Coleoptera), as it has been
suggested that DGCs may have evolved for different reasons in different
species (Chown and Nicholson,
2004; Chown et al.,
2006). Such acclimation studies will reveal whether or not other
factors, such as O2 or CO2, were important for the
evolution of DGCs in other species or whether in fact support is shown for a
single important evolutionary factor.

Finally, it is important that future research addresses the intriguing
findings of the present study. Cockroaches responded to all treatments by
altering their respiratory gas exchange patterns but the responses to
CO2 and O2 are not congruent with the predictions
stemming from the chthonic and oxidative damage hypotheses. Both the
CO2 and O2 responses are opposite to what is predicted.
Careful consideration needs to be given as to why the DGCs are responding to
these factors in this manner, and exploratory analyses of these new
observations might lead to new theories for the evolution of DGCs. It remains
to be seen if such theories supplant Buck, Keister and Specht's (Keister and
Specht, 1953) original hypothesis and the results of the present study, which
suggest that DGCs function to reduce respiratory water loss.

LIST OF ABBREVIATIONS

C

closed

DGC

discontinuous gas exchange cycle

F

flutter

O

open

PCO2

partial pressure of CO2

PO2

partial pressure of O2

RH

relative humidity

FOOTNOTES

We thank two anonymous referees for their helpful comments on an earlier
version of this manuscript. This research was supported by the
Australian Research Council (project number
DP0879605).

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JEB at SICB 2019

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Rasmus Ernhighlights a recent preprint from Takuya Sato and colleagues that investigates the effects of predation pressure and resource abundance on foraging behaviour of fish in social dominance hierarchies.